Non-Helicobacter pylori Helicobacter (NHPH) positive gastric cancer

Genetic analysis and culturing techniques for gastric non-Helicobacter pylori Helicobacter (NHPH) are progressing. NHPH is reported to accompany nodular gastritis, gastric MALT lymphoma, and mild gastritis. However, only a few gastric cancer cases infected by NHPH have been reported. PCR analysis specific for NHPH and H. pylori was performed for DNA from gastric mucosa of 282 Korean gastric cancer patients, who were treated with endoscopic submucosal dissection. For more precise strain detection of NHPH, NHPH-positive mucosa was stained by immunohistochemistry specific for Helicobacter suis. The Cancer Genome Atlas (TCGA) classification was analyzed for these 3 gastric cancer sub-groups by in situ hybridization and immunohistochemistry. Among 281 patients, 3 patients (1.1%) were positive for NHPH. One patient (Patient 1) was also positive for H. pylori by PCR, another patient (Patient 3) was positive for serum IgG for H. pylori, and the other patient (Patient 2) had no evidence for H. pylori infection. Gastric mucosa of Patients 2 and 3 were positive for H. suis staining. All three NHPH-positive gastric cancers were located in the antrum, and belonged to the Chromosomal Instability Type of TCGA classification. Gastric NHPH can be a cause of gastric cancer, although likely with lower pathogenesis than H. pylori.

In recent years, the importance of non-Helicobacter pylori Helicobacter (NHPH) is increasing due to advances in culturing techniques and genetic analysis for NHPH 1 . Eradication for Helicobacter pylori is also contributing to a focus on gastric diseases caused by NHPH. Gastric NHPH was originally reported as Gastrospirillum hominis in 1900 and subsequently renamed H. heilmannii [2][3][4] , having a larger body (5-6 μm × 0.5-0.6 μm) than H. pylori, with flagella on both sides. Gastric NHPH is reported to live in livestock and pets, and is found in 0.2-6% of gastric biopsies 5 . Gastric NHPH is associated with mild gastritis, nodular gastritis, and gastric mucosa associated lymphoid tissue (MALT) lymphoma [6][7][8] . However, there are only a few reports of NHPH associated with gastric cancer [9][10][11] .
In this study, we investigated the presence of NHPH in gastric mucosa in 281 Korean early gastric cancer patients, treated with endoscopic submucosal dissection (ESD). We found 3 gastric cancer patients with NHPH infection by NHPH-specific PCR. Clinicopathological features of these gastric cancers infected with NHPH will be presented. DNA extraction and PCR. DNA was extracted from four 10 µm thick paraffin sections for each noncancerous mucosa surrounding cancer with QIAamp DNA FFPE Tissue Kit (Qiagen Japan, Tokyo). All the extracted DNA was examined by real-time PCR for H. pylori and gastric NHPH using iQ™ SYBR Green supermix (Bio-Rad Laboratories). The real time PCR analysis was performed under the conditions of 5 min of preincubation at 95 °C, followed by 40 cycles of 30 s at 94 °C, 30 s at 60 °C, and 30 s at 72 °C. A final extension was performed for 7 min at 72 °C. And melting curve was confirmed with a stepwise increase in temperature from 55 to 95 °C in 0.5 °C/5 s increments. For the positive control for PCR, DNA extracted from H. suis infected mouse gastric sections was used, and for the negative control for PCR, DNA extracted from human gastric mucosal sections not infected with Helicobacter was used.  [13][14][15][16] . For H. pylori, the primers for the vacA gene were used. The PCR products for NHPH were purified from agarose gels using QIAquick Gel Extraction Kit (Qiagen Japan, Tokyo), and sequenced by Eurofins Genomics Japan.
Immunohistochemistry and in situ hybridization. We performed immunohistochemical analyses for NHPH-positive gastric cancers with anti-p53, E-cadherin (CDH1), and MLH1 antibodies, as well as in situ hybridization for Epstein-Barr virus-encoded small RNA (EBER), for the Cancer Genome Atlas (TCGA) classification 17,18 .
For the immunohistochemistry, histopathological specimens of the gastric cancers were deparaffinized by immersion in xylene solution (#241-00091, FUJIFILM Wako Pure Chemical Corporation, Japan) for 10 min at room temperature; then, antigen retrieval was performed by autoclave in citric acid buffer (pH = 6.0) (ab64214, abcam, UK) for 5 min at 121 °C. The slides were then immersed in 0.3% H 2 O 2 (#081-04215, FUJIFILM Wako Pure Chemical Corporation) / methanol (#137-01823, FUJIFILM Wako Pure Chemical Corporation) solution  For immunohistochemistry of H. suis, rabbit polyclonal antibody against H. suis was made. Briefly, a rabbit was immunized with a peptide expected to be antigenic from the vacA paralog reported in a literature 19 . Rabbit IgG was purified from the serum with Protein G affinity column. ELISA results for the specificity of this antibody are shown in Supplemental Fig. S1.
All methods were carried out in accordance with relevant guidelines and regulations.

Results
Patients. The characteristics of all 281 gastric cancer patients whose samples were obtained in this study were described in our previous study 12    There are no reported sequences that match the patient NHPH sequence, and it was also difficult to say the similarity, as the amplicon is too short. Of the three patients positive for NHPH, two were in group A (Patient 1 and Patient 2) and the other one was in group B (Patient 3). One patient in group A (Patient 2) was negative for the H. pylori-specific vacA gene on PCR and may have only had infection with NHPH. The other patient in group A (Patient 1) was positive for the H. pylori-specific vacA gene by PCR, even though serum H. pylori IgG was negative, and had co-infection of NHPH and H. pylori. The group B patient (Patient 3) was negative for the H. pylori-specific vacA gene on PCR and had no medical history of eradication therapy. Table 2 describes the main findings for the 3 patients positive for NHPH.

NHPH immunohistochemistry.
As PCR for the precise strain of NHPH was negative for the NHPH positive patients, we performed immunohistochemistry for H. suis, which is reported to be the most prevalent NHPH for human stomach 19,20 . H. suis was strongly positive in Patient 2, weakly positive in Patient 3 and negative in Patient 1 (Fig. 2). Thus, the NHPH in Patients 2 and 3 appeared to be H. suis. The positive staining of H. suis in Patient 2 was located deep in the gastric glands, as well as in surface epithelium. TCGA classification. For specifying gastric cancer under infection of NHPH, we determined the TCGA classification of these three gastric cancers with immunohistochemistry for MLH-1, p53, and CDH-1, and EBER in situ hybridization. All three gastric cancer were well-differentiated adenocarcinoma, MLH-1 positive, p53-positive, CDH-1-positive, and EBER-negative, leading to classification as in the Chromosomal Instability (CIN) Type (Fig. 4).

Discussion
We identified three NHPH positive patients with gastric cancer treated by endoscopic resection. Two of them showed co-infection with H. pylori, but the remaining patient had no evidence of H. pylori infection.
Gastric NHPH is reported to cause gastric MALT lymphoma and nodular gastritis with mild atrophy in the corpus 21 . However, the number of reports of gastric cancer associated with NHPH infection is very small 9-11 . Nogueira's case was co-infected with H. pylori and no precise characteristics of gastric cancer and gastritis in this patient were reported 10 . Yang's case did not check for H. pylori infection 11 . This patient had intestinal-type  www.nature.com/scientificreports/ infection. However, Kimura-Takemoto classification of the gastric mucosa of Patient 2 was C-II, and thus natural disappearance of H. pylori is less likely. Patient 2 may really be negative for H. pylori. Therefore, in this patient, NHPH is speculated to have pathogenicity for gastric cancer. This is the first case of gastric cancer with NHPH infection, confirmed without infection of H. pylori.
We have previously shown that Trefoil Factor Family 3 (TFF3), a small peptide of 12-20 kD with a trefoil motif, which is secreted from various mucus-secreting cells, is an effective serum biomarker for gastric cancer both in Japan and in South Korea 12 . Although the mechanism of elevated serum TFF3 in cancer patients is not  The origin of NHPH infection in these 3 patients is not known. The patients were not keeping pets, and were not working in animal handling 23,24 . The patients might have been infected with NHPH through food without proper cooking. H. suis is highly prevalent in pig stomach (60-80%) 20 . NHPH is lacking in a Cag pathogenicity island and would be less pathogenic than H. pylori . As to the virulence factor of H. suis, we would suggest HsvA protein, used in the immunohistochemical study, as one of the candidates because of its similarity to H. pylori autotransporter proteins ImaA, FaaA, and VlpC.
The presence of gastric cancer in the patient with NHPH single infection suggests that eradication of NHPH for preventing gastric cancer may be merited. NHPH can be eradicated by the same drugs used for H. pylori 6,[13][14][15] . However, the urease activity of NHPH depends on the strain and NHPH single infection would show as rapid urease test negative, serum H. pylori IgG negative, stool H. pylori antigen negative, and urea breath test negative 6,15,16 . For the diagnosis of NHPH, it is important to consider the NHPH infection in patients without atrophic gastritis and negative for H. pylori. Diagnostic systems for NHPH infection are now under development.
In conclusion, we identified three patients with gastric cancer who were positive for NHPH, including one patient was without co-infection with H. pylori. NHPH could be one of the causes for gastric cancer in Korea and partially explain differences between serum TFF3 and ABCD classifications between Korea and Japan. NHPH eradication could be also important to prevent gastric carcinogenesis in this H. pylori eradication era.